Mechanism of mitochondrial membrane permeabilization by HIV-1 Vpr, mimetics of Vpr and methods of screening active molecules having the ability to alter and/or prevent and/or mimic the interaction of Vpr with ANT

ABSTRACT

The invention is directed to the induction of mitochondrial membrane permeabilization via the physical and functional interaction of the HIV-1 Vpr protein with the mitochondrial inner membrane protein ANT (adenine nucleotide translocator, also called adenine nucleotide translocase or ADP/ATP carrier). Reagents and methods for inducing and/or inhibiting the binding of Vpr to ANT, mitochondrial membrane permeabilization, and apoptosis are provided.

[0001] This application claims the benefit of U.S. ProvisionalApplication Ser. No. 60/231,539, filed Sep. 11, 2000, and of U.S.Provisional Application Ser. No. 60/232,841, filed Sep. 15, 2000, bothof which are hereby incorporated by reference.

DESCRIPTION OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The invention is directed to discovery that the proapoptoticHIV-1-encoded protein Vpr induces mitochondrial membranepermeabilization via its physical and functional Interaction with themitochondrial Inner membrane protein ANT (adenine nucleotidetranslocator, also called adenine nucleotide translocase or ADP/ATPcarrier). HIV-1 Viral protein R (Vpr) interacts with the permeabilitytransition pore complex (PTPC) to trigger ANT pore formation and/ormitochondrial membrane permeabilization (MMP) and consequent cell death(by apoptosis or any related mechanism of cell death).

[0004] 2. Background of the Invention

[0005] It is now recognized that mitochondria play an important role incontrolling the life and death (the apoptosis) of cells (Kroemer andReed 2000). Thus it seems that a growing number of molecules areinvolved in signal transduction, and that many metabolites (and certainviral effectors) act on the mitochondria and influence thepermeabilization of mitochondrial membranes. Also, a certain number ofexperimental anti-cancer drugs kill cells by acting directly onmitochondrial membranes (Ravagnan et al., 1999; Larochette et al., 1999;Marchetti et al., 1999; Fulda et al., 1999; Belzacq et al., 2000).Therefore, the use of specific pro-apoptotic agents for mitochondriaseems to be a concept that is emerging in anti-cancer chemotherapy (forreference: Costantini, et al., 2000). A possible outcome could be theuse of cytoprotective molecules to treat illnesses associated withexcess apoptosis (AIDS, neurodegenerative diseases, etc.) owing to theirability to stabilize mitochondrial membranes. Against this background,the identification (mode of action) of those molecular components thatcontrol the permeability of the mitochondrial membranes has become amajor topic in biomedicine.

[0006] MMP is a key event of apoptotic cell death associated with therelease of caspase activators and caspase-independent death effectorsfrom the intermembrane space, dissipaton of the inner transmembranepotential (ΔΨm), as well as a perturbation of oxidative phosphorylationG. Kroemer, N. Zamzami, S. A. Susin, Immunol. Today 18, 44-51 (1997). D.R. Green, J. C. Reed, Science 281, 1309-1312 (1998). J. J. Lemasters, etal., Biochim. Biophys. Acta 1366, 177-196 (1998). D. C. Wallace, Science283, 1482-1488 (1999). M. G. Vander Heiden, C. B. Thompson, Nat. CellBiol. 1, E209-E216 (1999). A. Gross, J. M. McDonnell, S. J. Korsmeyer,Genes Dev. 13, 1988-1911 (1999). G. Kroemer, J. C. Reed, Nat. Med. 6,513-519 (2000). Pro- and anti-apoptotic members of the Bcl-2 familyregulate inner and outer MMP through interactions with the adeninenucleotide translocator (ANT; in the inner membrane, IM), thevoltage-dependent anion channel (VDAC; in the outer membrane, OM) and/orthrough autonomous channel-forming activities G. Kroemer, N. Zamzami, S.A. Susin, Immunol. Today 18, 44-51 (1997). D. R. Green, J. C. Reed,Science 281, 1309-1312 (1998). J. J. Lemasters, et al., Biochim.Biophys. Acta 1366, 177-196 (1998). D. C. Wallace, Science 283,1482-1488 (1999). M. G. Vander Heiden, C. B. Thompson, Nat. Cell Biol.1, E209-E216 (1999). A. Gross, J. M. McDonnell, S. J. Korsmeyer, GenesDev. 13, 1988-1911 (1999). G. Kroemer, J. C. Reed, Nat. Med. 6, 513-519(2000). I. Marzo, et al., Science 281, 2027-2031 (1998). S. Shimizu, M.Narita, Y. Tsujimoto, Nature 399, 483-487 (1999). S. Shimizu, A.Konishi, T. Kodama, Y. Tsujimoto, Proc. Natl. Acad. Sci. USA 97,3100-3105 (2000). S. Desagher. et al., J. Cell Biol. 144, 891-901(1999).

[0007] ANT and VDAC are major components of the permeability transitionpore complex (PTPC), a polyprotein structure organized at sites at whichthe two mitochondrial membranes are apposed. G. Kroemer, N. Zamzami, S.A. Susin, Immunol. Today 18, 44-51 (1997). D. R. Green, J. C. Reed,Science 281, 1309-1312(1998). J. J. Lemasters, et al., Biochim. Biophys.Acta 1366, 177-196 (1998). D. C. Wallace, Science 283, 1482-1488 (1999).M. G. Vander Heiden, C. B. Thompson, Nat. Cell Biol. 1, E209-E216(1999). A. Gross, J. M. McDonnell, S. J. Korsmeyer, Genes Dev. 13,1988-1911 (1999). G. Kroemer, J. C. Reed, Nat. Med. 6, 513-519 (2000).M. Crompton, Biochem. J. 341, 233-249 (1999).

[0008] The adenine nucleotide translocator (ANT) plays an important rolein the process that triggers the permeabilization of mitchondrialmembranes, and subsequent apoptosis (Marzo, et al., 1998; Brenner, etal., 2000). In the cellular context, ANT Is inserted into the internalmembrane of mitochondria and has two opposing functions. On the onehand, ANT is a vital antiport for cellular bioenergetics and is specificto ATP and ADP. On the other hand, ANT can form a non-specific lethalpore through the action of certain ligands (natural or xenobiotic) thateliminate the mitochondrial electrochemical gradient.

[0009] The HIV-1 regulatory protein Vpr has pleiotropic effects on viralreplication and cellular proliferation, differentiation, cytokineproduction, and NF-kB-mediated transcription. M. Emerman, M. H. Malim,Science 280, 1880-1884 (1998). A. D. Frankel, J. A. T. Young, Annu. Rev.Biochem. 67, 1-25 (1998). M. Bukrinsky, A. Adzhubei, J. Med. Virol 9,39-49 (1999). In addition, Vpr can localize to mitochondria. I. G.Macreadie, et al., Proc. Natl. Acad. Sci. USA 92, 2770-2774 (1995). I.G. Macreadie, et al., FEBS Lett. 410, 145-149 (1997). K. Muthami, L. J.Montaner, V. Ayyavoo, D. B. Weine. DNA and Cell Biology 19, 179-188(2000). E. Jacotot, et al., J. Exp. Med. 191, 33-45 (2000). Full length(Vpr1-96) or truncated synthetic forms of Vpr act on the PTPC to induceall mitochondrial hallmarks of apoptosis, including ΔΨ_(m) loss and therelease of cytochrome c and apoptosis inducing factor (AIF). E. Jacotot,et al., J. Exp. Med. 191, 33-45 (2000). The MMP-inducing activity of Vprresides in its C-terminal moiety (Vpr52-96), within an α-helical motifof 12 amino acids (Vpr71-82) containing several critical arginine (R)residues (R73, R77, R80) which are strongly conserved among differentpathogenic HIV-1 isolates. L. G. Macreadie, et al., Proc. Natl. Acad.Sci. USA 92, 2770-2774 (1995). I. G. Macreadie, et al., FEBS Lett. 410,145-149 (1997). E. Jacotot, et al., J. Exp. Med. 191, 33-45 (2000).

[0010] Depending on the apoptotic stimulus, permeabilization may affectthe OM and IM in a variable fashion and may or may be not accompanied bymatrix swelling. G. Kroemer, N. Zamzami, S. A. Susin, Immunol. Today 18,44-51 (1997). D. R. Green, J. C. Reed. Science 281, 1309-1312 (1998). J.J. Lemasters, et al., Biochim. Biophys. Acta 1366, 177-196 (1998). D. C.Wallace, Science 283, 1482-1488 (1999). M. G. Vander Heiden, C. B.Thompson, Nat. Cell Biol. 1, E209-E216 (1999). A. Gross, J. M.McDonnell, S. J. Korsmeyer, Genes Dev. 13, 1988-1911 (1999). G. Kroemer,J. C. Reed. Nat. Med. 6, 513-519 (2000). In vitro experiments performedon purified mitochondria or proteins reconstituted into artificialmembranes suggest at least two competing models of MMP. On the one hand,pore formation by ANT has been proposed to account for IMpermeabilization, osmotic matrix swelling, and consequent OM rupture,resulting because the surface area of the IM with its folded christaeexceeds that of the OM. In support of this hypothesis, pro-apoptoticmolecules such as Bax, atractyloside, Ca²⁺, and thiol oxidants cause ANT(which normally is a strictly specific ADP/ATP antiporter) to form anon-specific pore (I. Marzo, et al., Science 281, 2027-2031 (1998); N.Brustovetsky, M. Klingenberg, Biochemistry 35, 8483-8488 (1996); C.Brenner, et al., Oncogene 19, 329-336 (2000)). On the other hand, VDAChas been suggested to account for a primary OM permeabilization notaffecting IM (S. Shimizu, M. Narita, Y. Tsujimoto, Nature 399, 483-487(1999). S. Shimizu, A. Konishi, T. Kodama, Y. Tsujimoto, Proc. Nat.Acad. Sci. USA 97, 3100-3105 (2000)). In favor of this hypothesis, thepermeabilization of VDAC-containing liposomes to sucrose or cytochrome cis enhanced by Bax and inhibited by Bcl-2 in vitro. S. Shimizu, M.Narita, Y. Tsujimoto, Nature 399, 483-487 (1999). S. Shimizu, A.Konishi. T. Kodama, Y. Tsujimoto, Proc. Natl. Acad. Sci. USA 97,3100-3105 (2000).

[0011] Recent studies have revealed the existence of several viralapoptosis inhibitors acting on mitochondria. For example, adenovirus,Epstein Barr virus, Herpes virus saimiri, and Kaposi sarcorma-associatedhuman herpes virus 8 produce apoptosis-suppressive Bcl-2 homologs. E.H.-Y. Cheng, et al., Proc. Natl. Acad. Sci. USA 94, 690-694 (1997). J.H. Han, D. Modha, E. White, Oncogene 17, 2993-3005 (1998). T. Derfuss,et al., J. Virol. 72, 5897-5904 (1998). W. L. Marshall, et al., J.Virol. 73, 5181-5185 (1999). In addition, several viruses encodePTPC-interacting proteins without any obvious homology to the Bcl-2/Baxfamily. The cytomegalovirus apoptosis inhibitor pUL37x (V. S.Goldmacher, et al., Proc. Natl. Acad. Sci. USA 96, 12536-12541 (1999).)and Vpr, an HIV-1-encoded apoptosis inducer, selectively bind to ANT.The proapoptotic p13 (II) protein derived from the X-II ORF of HTLV-1 isalso targeted to mitochondria via a peptide motif that bears structuralsimilarities to the mitochondriotoxic domain of Vpr. V. Ciminale, etal., Oncogene 18, 4505-4514 (1999). Moreover, the pro-apoptoticMMP-inducing hepatitis virus B protein X interacts with VDAC. Z Rahmani,K. W. Huh, R. Lasher, A. Siddiqui, J. Virol. 74, 2840-2846 (2000). Thus,both VDAC and ANT emerge as major targets of viral apoptosis regulationand, perhaps, as targets for pharmacological intervention on viralpathogenesis and/or other pathologies linked to apoptosis dysregulations(i.e., cancer, ischemia, neurodegenerative diseases, etc.). Apoptosis isa process that develops in several phases: (1) an initiation phase,which is extremely heterogeneous and during which the biochemicalpathways paticipating in the process depend on the apoptosis-inducingagent; (2) a decision phase, which is common to different types ofapoptosis, during which the cell “decides” to commit suicide; and (3) acommon degradation phase, which is characterized by the activation ofcatabolic hydrolases (caspases and nucleases). Although the activationof caspases (cysteine proteases cleaving at aspartic acid [Asp]residues) and nucleases is necessary for the acquisitions of the fullapoptotic morphology, it appears clear that inhibition of such enzymesdoes not inhibit cell death induced by a number of different triggers:Bax, Bak, c-Myo, PML, FADD, glucocorticoid receptor occupancy, tumornecrosis factor, growth factor withdrawal, CXCR4 cross-linking, andchemotherapeutic agents, such as etoposide, camptothecin, or cisplatin.In the absence of caspase activation, cells manifest a retardedcytolysis without characteristics of advanced apoptosis, such as totalchromatin condensation, oligonucleosomal DNA fragmentation, andformation of apoptotic bodies. However, before cells lyse, they domanifest a permeabilization of both mitochondrial membranes withdissipation of the inner transmembrane potential (ΔΨ_(m)) and/or therelease of apoptogenic proteins, such as cytochrome c andapoptosis-inducing factor (AIF) via the outer membrane. These resultshave invalidated the hypothesis that caspase activation is alwaysrequired for apoptotic cell death to occur. Rather, cell death isintimately associated with the permeabilization of mitochondrialmembranes.

[0012] The understanding of apoptosis has recently been facilitated bythe development of cell-free Systems. Instead of considering the cell asa black box, subcellular fractions (e.g., mitochrondria, nuclei, andcytosol) are mixed together with the aim to reconstitute the apoptosisphenomenon by recapitulating the essential steps of the process invitro. It appears that proapoptotic second messengers, whose naturedepends on the apoptosis-inducing agent, accumulate in the cytosolduring the initiation phase. These agents then induce mitochondrialmembrane permeabilization, allowing cells to enter the decision phase.The apoptotic changes of mitochondria consist in a ΔΨ_(m) loss,transient swelling of the mitochondrial matrix, mechanical rupture ofthe outer membrane and/or its nonspecific permeabilization by giantprotein-permanent pores, and release of soluble intermembrane proteins(SIMPs) through the outer membrane. Once the mitochondrial membranebarrier function is lost, several factors, e.g., the metabolicconsequences at the bioenergetic level, the loss of redox homeostasis,and the perturbation of ion homeostais, contribute to cell death. Theactivation of proteases (caspases) and nucleases by SIMP's is necessaryfor the acquisition of apoptotic morphology. This latter phasecorresponds to the degradation step, beyond the point of no return ofthe apoptotic process. Different SIMPs provide a molecular link betweenmitochondrial membrane permeabilization and the activation of catabolichydrolases: cytochrome c (a heme protein that participates in caspaseactivation), certain procaspases (in particular, procaspases 2 aid 9,which in some cell types, are selectively enriched in mitochondria), andAIF, AIF is a nuclear-encoded intermembrane flavoprotein thattranslocates to the nucleus where it induces the caspases-independentperipheral chromatin condensation and the degradation of DNA into50-kilobase pair fragments.

[0013] The mechanism of mitochondrial membrane permeabilization is notcompletely understood. Some investigators prefer the hypothesis thatproapoptotic members of the Bcl-2 family are inserted in the outermembrane where they oligomerize and form cytochrome c permeant pores inan autonomous fashion, not requiring the interaction with othermitochondrial membrane proteins. However, Bax-induced membranepermeabilization is inhibited by cyclosporin A (CsA) and bongkrekic acid(BA), two inhibitors of formation of the permeability transition pore(or “megachannel”), suggesting that sessile mitochrondrial proteins (thetargets of CsA and BA) are involved in this process. The permeabilitytransition pore has a polyprotein structure that is formed at thecontact sites between the inner and outer membranes. One of the keyproteins of the permeability transition pore complex (PTPC) is theadenine nucleotide translocator (ANT). ANT, the target of BA, is themost abundant inner membrane protein, ANT normally functions as aspecific carrier protein for the exchange of adenosine triphosphate(ATP) and adenosine diphosphate (ADP), but it can become a nonspecificpore.

[0014] An interesting property of the PTPC is that the permeabilizationof the inner and/or outer mitochondrial membranes compromises thebioenergetic equilibrium of the cell (e.g., it provokes the oxidation ofreduced NADPH and glutathione, the depletion of ATP, and the dissipationof ΔΨ_(m)and effects the homeostasis of intracellular ions (e.g. byreleasing Ca²⁺ from the matrix). Intriguingly, all of these changesthemselves increase the probability of PTPCs opening. This has twoimportant implications. First, the consequences of PTPC openingthemselves favor opening of the PTPC in a self-amplification loop thatcoordinates the lethal response among mitochondria within the samecells. Second, this implies that the final result of PTPC opening is astereotyped ensemble of biochemical alterations, which does not dependon the initiating stimulus, be it a specific proapoptotic signaltransduction cascade or nonspecific damage at the energy or redoxlevels.

[0015] Chemotherapy aims at the specific eradication of cancer cells,mostly through the induction of apoptosis. Gene therapy can employBax-delivering vectors, thereby indirectly targeting mitochondria toinduce apoptosis. In contrast to such proteins, certain peptides readilypenetrate the plasma membrane and thus can be used as the pharmacologicagents. Mastoparan, a peptide isolated from wasp venom, is the firstpeptide known to induce mitochondral membrane permeabilization via aCsA-inhibitable mechanism and to induce apoptosis via a mitochondrialeffect when added to intact cells. This peptide has an α-helicalstructure and possesses some positive charges that are distributed onone side of the helix. A similar peptide (KLAKLAKKLAKLAK or (KLAKLAK)₂(K=lysine, L=amine, and A=leucine) has been found recently to disruptmitochondral membranes when it is added to purified mitochondria,although the mechanisms of this effect have not been elucidated.(Ellerby, H. M. et al., Anti-cancer activity of targeted pro-apoptoticpeptides, Nature Med. 5, 1032-1038 (1999)).

[0016] The proapoptotic 96 amino acid protein viral protein R (Vpr) fromhuman immunodeficiency virus-I contains a comparable structural motif(aa 71-82), i.e., an α-helix with several cationic charges thatconcentrate on the same side of the helix. Vpr, as well as Vprderivatives containing this “mitochondriotoxic” domain cause a rapid CsAand BA-inhibited dissipation of the ΔΨ_(m) as well as the mitochondrialrelease of apoptogenic proteins, such as cytochrome c or AIF. The samestructural motifs relevant for cell killing appear to be responsible forthe mitochondriotoxic effects of Vpr. Vpr favors the permeabilization ofartificial membranes containing the purified PTPC or defined PTPCcomponents such as the ANT combined with Bax, but this effect isprevented by the addition of recombinant Bcl-2. According to surfaceplasmon resonance studies, the Vpr C-terminus binds purified ANT with ahigh affinity in the nanomolar range. E. Jacotot et al., J. Exp. Med.191, 33-45 (2000), which is specifically incorporated herein byreference. In addition, a biotinylated Vpr-derived peptide (Vpr52-96)may be employed as bait to specifically purify a mitochondrial molecularcomplex containing ANT and the VDAC. Yeast strains lacking ANT or VDACare less susceptible to Vpr-induced killing than are control cells.Thus, Vpr induces apoptosis via a direct effect on the mitochondrialPTPC. In analogy to Vpr, the p13 (II) protein derived from the X-II openreading frame of HTLV-1 is targeted to mitochondria and can cause adissipation of the ΔΨ_(m) and mitochondrial swelling. Mitochondrialtargeting of this protein has been mapped to a decapeptide sequence thatcontains several Arg residues that are asymmetrically distributed in theα-helix. However, Arg-Ala substitutions within the mitochondriotoxicdomain of p13 (II) did not abolish the mitochondrial targeting of p13.

[0017] Lethal peptides may be targeted to mitochondria and morespecifically, at least in the case of Vpr, to the PTPC. Ellerby et al.recently have fused the mitochondriotoxic (KLAKLAK)₂ motif to atargeting peptide that interacts with endothelial cells. Such a fusionpeptide is internalized and induces mitochondrial membranepermeabilization in angiogenic endothelial cells and kills MDA-MD-435breast cancer xenografts transplanted into nude mice. Similarly, arecombinant chimeric protein containing interleukin 2 (IL-2) proteinfused to Bax selectively binds to and kills IL-2 receptor-bearing cellsin vitro. Thus, specific cytotoxic agents that target surface receptors,translocate into the cytoplasm, and induce apoptosis via mitochondrialmembrane permeabilization might be useful in treating cancer.

[0018] A recurrent problem with conventional chemotherapeutic agents isthat they exploit endogenous apoptosis-induction pathways that may becompromised by alterations such as mutations of p53, increasedantioxidant activity, blockade of CD95/CD95L pathway, overexpression ofBcl-2-like proteins, etc. One possible strategy to enforce cell death isto trigger downstream events of the common apoptotic pathway. Thus,adenovirus-mediated transfer of caspases has been proposed as onestrategy to induce cell death beyond any regulation. An alternativestrategy is to use mitochondriotoxic agents that induce cell deathirrespective of the upstream control mechanisms and irrespective of thestatus of caspases and endogenous caspase inhibitors. As an example,LND, arsenite, or CD437 induce cell death independently of the p53status via a pathway that is not affected by caspase inhibitors.Similarly, betulinic acid and Vpr trigger CD95 (Apo-1/Fas)- andp53-independent apoptosis, and both permeabilize mitochondrial membranesin a caspase-independent fashion. As a result, these types of agents mayprove to be highly useful in killing normally resistant cells. Moreover,the future of tumor therapy may profit from the design of agents thatovercome the Bcl-2-mediated stabilization of mitchondrial membranes aswell as from targeting amphipathic peptides or peptidomimetics todefined cellular populations or tissues.

[0019] Selective eradication of transformed cells by use ofmitochondrion-specific agents should be effective. One strategy is totarget a toxic agent to selected cell types on the basis of the specificexpression of surface receptors. Another, yet to be developed, strategywould aim at exploiting difference in the composition or regulation ofthe PTPC between normal and tumor cells. Future research will tell towhich extent cell targeting (by use of retroviral or adenoviral vectors,use of integrin-specific domains, etc.) and/or targeting oftumor-specific alterations in the PTPC will prove to be useful in cancertherapy, and also in the treatment of neurodegenerative diseaseshypothetically linked to mitochondrial dysfunction (i.e., Friedrichataxia, Hereditary spastic paraplegia, Huntington disease, Amyotrophiclateral sclerosis, Parkinson disease, Alzheimer disease) and treatmentof acute organ failure that may involve regulatory events acting at thelevel of MMP (i.e., ischemia) (Kroemer, G. et al., Mitchondrial controlof cell death, Nature Med., vol. 6, no. 5, 513-519 (1999)).

[0020] Thus, there exist a need in the art for methods and reagents forregulating mitochondrial permeabilization and apoptosis.

SUMMARY OF THE INVENTION

[0021] The present invention relates to the physical and functionalinteractions between Vpr and the adenine nucleotide translocator (ANT),which function to permeabilize mitochondrial membranes and result in thedeath of cells by apoptosis. In a preferred embodiment, the presentinvention relates to the physical and functional interactions betweenVpr and the three human isoforms of ANT also designated ANT1, ANT2, andANT3. The invention encompasses methods of exploiting this novelmechanism to permeabilize mitochondrial membranes. The invention furtherencompasses methods of causing cell death by apoptosis.

[0022] The invention also encompasses methods of altering or preventingbinding of Vpr to ANT. The invention further encompasses methods ofaltering or preventing channel formation due to the association of Vprwith ANT. The invention also encompasses methods of causing orpreventing permeabilization of mitchondrial membranes. The inventionalso encompasses methods of causing or preventing cell death byapoptosis.

[0023] The invention also encompasses methods of screening for moleculesthat alter or prevent binding of Vpr to ANT. The invention furtherencompasses methods of screening for molecules that alter or preventchannel formation due to the association of Vpr with ANT. The inventionalso encompasses methods of screening for molecules that cause orprevent permeabilization of mitochondrial membranes. The invention alsoencompasses methods of screening for molecules that cause or preventcell death by apoptosis.

[0024] The invention also encompasses methods of screening for moleculesthat compete with the binding of the C-terminal moeity of Vpr (vpr52-96for HIV-1) to ANT. The invention also encompasses methods of screeningfor molecules that promote the binding of the C-terminal moeity of Vpr(vpr52-96 for HIV-1) to ANT. The invention also encompasses methods ofscreening for molecules that alter or prevent binding of the C-terminalmoeity of Vpr (vpr52-96 for HIV-1) to ANT. The invention furtherencompasses methods of screening for molecules that alter or preventpermeabilization of mitochondrial membranes due to the association ofthe C-terminal moeity of Vpr (vpr52-96 for HIV-1) with ANT. Theinvention further encompasses methods of screening for molecules thatalter or prevent apoptosis due to the association of the C-terminalmoeity of Vpr (vpr52-96 for HIV-1) with ANT.

[0025] The invention also encompasses peptidic or non-peptidic moleculesthat alter or prevent binding of Vpr to ANT. The invention alsoencompasses peptidic or non-peptidic molecules that mimic Vpr or Vprfragment in its capacity to interact physically or functionally withANT. The invention further encompasses peptidic or non-peptidicmolecules that alter or prevent channel formation due to the associationof Vpr with ANT. The invention also encompasses peptidic or non-peptidicmolecules that cause or prevent permeabilization of mitochondrialmembranes. The invention also encompasses peptidic or non-peptidicmolecules that cause or prevent cell death by apoptosis. The inventionfurther encompasses pharmaceutical and diagnostic compositionscomprising these molecules and the use of these compositions to cause orprevent permeabilization of mitochondrial membranes or apopotosis.

[0026] The invention further encompasses peptidic or non-peptidicmolecules that mimic the C-terminal moeity of Vpr (vpr52-96 for HIV-1)and modulate the permeabilization of mitochondrial membranes. Theinvention also encompasses peptidic or non-peptidic molecules thatcompete with the binding of the C-terminal moeity of Vpr (vpr52-96 forHIV-1) to ANT. The invention also encompasses peptidic or non-peptidicmolecules that promote the binding of the C-terminal moeity of Vpr(vpr52-96 for HIV-1) to ANT. The invention further encompassespharmaceutical and diagnostic compositions comprising these moleculesand the use of these compositions to cause or prevent permeabilizationof mitochondrial membranes or apopotosis.

[0027] The invention also encompasses methods for screening for geneticor epigenetic alterations in the expression or structure of the threeANT isoforms in humans. The invention further encompasses screening anddiagnosis for differences in the ability of the three ANT isoforms indifferent patients to interact with Vpr and to promote mitochondrialmembrane permeabilization, channel formation and/or apopotosis.

[0028] The invention also encompasses methods for specific cell killingby induction of apoptosis.

[0029] The invention also encompasses methods for screening moleculesmodifying channel properties of ANT.

[0030] The invention further encompasses methods of screening of activemolecules able to alter or prevent ANT-Bcl2 interaction.

[0031] By studying the cytotoxic properties of the Vpr protein of HIV-1,the inventors discovered that Vpr interacts direct with ANT to triggerthe permeabilization of mitochondrial membranes, as well as apoptosis.First, Vpr goes through an external mitochondrial membrane using themitchondrial protein (also called “voltage-dependent anion channel”:VDAC) and then attaches itself to the ANT, its primary target, withstrong affinity (KD=1 nM). The ANT/Vpr complex forms high-conductancechannels that trigger the permeabilization of the internal mitochondrialmembrane, the swelling of the mitochondrial matrix and, finally, thebreakage of the external membrane, and thus the release of factors thatimplement apoptosis (e.g., AIF, cytochrome c and some pro-caspases). Theinventors have identified interaction sites between ANT and Vpr: for Vpr(14 Kd; 96 aa), the binding site to ANT brings into play the pattern71HFRIGCRHSRIG82 (minimal toxic pattern) in the heart of the linearstructure (α-helicoidal between amino-acids 52 and 83) of Vpr 52-96. ForANT1 (30 Kd; 298 aa), the binding site to Vpr brings into play thepattern 104DRHKQFWRYFAGN116 in the middle of the second ANT ring (aa92-116).

[0032] The inventors' discovery of the physical and functionalinteraction between Vpr and ANT, and of at least one of the interactionsites, led them to build analogs of said toxic pattern of Vpr that caninteract with the protein complex (permeability transition pore; PTPC)that contains ANT. These molecules can serve to imitate thepro-apoptotic effect of Vpr in order to destroy cancerous cells in vitroor in vivo. These molecules are either peptide or non-peptide moleculesand are acquired in isolated or purified form.

[0033] The present invention pertains to a novel protein/proteininteraction between the retroviral HIV regulatory protein Vpr and themitochondrial adenine nucleotide translocator (ANT) a membraneassociated receptor implicated in the control of cell death byapoptosis. The invention also concerns peptidic or non-peptidicmolecules having the ability to alter and/or to prevent the binding (orthe chanel formation due to this binding) of Vpr to ANT. Another aspectof the invention concern peptidic or non-peptidic molecules having theability to mimic the C-terminal moiety of Vpr (Vpr52-96 for HIV-1) inits capacity to bind ANT and cooperate with ANT to permeabilisemitochondrial membranes (and consequently kill cells). The invention isalso directed to pharmaceutical and diagnostic compositions containingan effective amount of the molecules altering and/or preventing thebinding (and/or conformational consequences of this binding such aschanel formation) of Vpr to ANT (consequently such compositions will becytoprotectives), as well as to therapeutic or diagnostic methods usingsuch pharmaceutical or diagnostic composition. Moreover, the inventionis also directed to pharmaceutical and diagnostic compositionscontaining an effective amount of the molecules able to mimic theC-terminal part of Vpr in its capacity to bind and cooperate with ANT topermeabilise mitochondrial membranes (and consequently kill cells), aswell as to therapeutic or diagnostic methods using such pharmaceuticalor diagnostic composition. The invention also deals with methods ofscreening new active molecules (endogenous or xenobiotics) having theability to alter and/or to prevent the binding (or the chanel formationdue to this binding) of Vpr to ANT, or having the ability to mimic theC-terminal moiety of Vpr (Vpr52-96 for HIV-1) in its capacity to bindANT and cooperate with ANT to permeabilise mitochondrial membranes (andconsequently kill cells). Finally the invention is directed to methodsof screening genetics or epigenetics (such as specific modifications incancer affected individuals) alterations in the expression or structureof the three ANT isoforms in humans.

[0034] Thus, the present invention concerns a protein-to-proteininteraction between Vpr and ANT, and potentially between Vpr and VDAC,which can exploited to screen therapeutic molecules active ascytoprotectors (an inhibitor of ANT/Vpr interaction) or active ascytotoxics (analogs of Vpr with respect to interaction with ANT and/orVDAC). In this regard, the inventors have established a new ELISAscreening test for Vpr ligands and molecules than can inhibit theattachment of ANT to Vpr.

[0035] Consequently, one of the objectives of the present inventionconcerns peptide or non-peptide molecules that can imitate Vpr byattaching themselves to ANT in the cells (or certain cells) of anindividual; specifically, a person afflicted with cancer.

[0036] The invention also concerns structural or functional inhibitorseffective in blocking Vpr/ANT interaction or Vpr/VDAC interaction andthus 1) that inhibit in vitro or in vivo infection by HIV and 2) thatinhibit the cytotoxic effect of any ANT ligand (natural, endogenous,xenobiotic) and thus produce a cytoprotective effect in patientsafflicted with a disease associated with excess apoptosis.

[0037] Thus, the present invention also covers components that canmodify the interaction between, on the one hand, Vpr (found in thecells, extracellular fluids, or HIV particles of an individual infectedby a retrovirus) or an analog (endogenous [e.g., Bcl-2 or a sub-regionof this protein] or xenobiotic) of Vpr and, on the other hand, at leastone of the isoforms of ANT found in cells at the mitochondrial membranelevel. Molecules derived from ANT or from an interaction pattern (forexample, ANT104-116) with Vpr are also considered as active moleculesforming a part of the present invention.

[0038] The invention also concerns the use of the compounds andinhibitors defined earlier as active principles of pharmaceuticalcompounds. One possible specific application might be the coupling ofVpr, from a Vpr pattern (e.g., pattern 52-96, 71-82, or 71-96) or froman analog of Vpr, with a molecule that can screen a tumor in vivo. Thus,the invention includes the use of a Vpr pattern (for example, pattern52-96, 71-82, or 71-96).

[0039] The invention also includes the means to screen molecules thatcan imitate the cytotoxic and/or mitochondrial effect of Vpr(particularly its interaction with ANT and/or VDAC) and the means toscreen molecules (cytoprotective) that can modify the interactionbetween, on the one hand, Vpr (found in the cells, extra-cellularfluids, or HIV particles of individuals infected by a retrovirus) or ananalog (endogenous or xenobiotic) of Vpr and, on the other hand, atleast one of the isoforms of ANT found in the cell at the mitochondrialmembrane level.

[0040] Thus, the invention includes the methods to screen agonists(structural or functional analogs of Vpr) or antagonists (inhibitors ofVpr/ANT interaction, or the adoption of a “lethal pore” conformation inresponse to Vpr, or a structural or functional analog of Vpr) of ANT.Hence, the invention includes at least two screening tests:

[0041] a binding test of ANT (or an ANT derived peptide containing, forexample, the pattern 104-116 of ANT) on Vpr (or a peptide derived fromVpr containing, for example, the pattern 71-82). The protocol of thistest has already been established in the case of binding Vpr 52-96 atthe bottom of a plate with 96 wells and to which an ANT or a peptide ofANT containing the pattern 104-116 is attached and then exposed.

[0042] a test called a “double test” functional for ANT and thatsimultaneously evaluates the specific antiport function and thenon-specific lethal pore function of ANT. The principle of this test andthe detailed protocol are described in Example 5.

BRIEF DESCRIPTION OF THE DRAWINGS

[0043]FIG. 1 presents physical and functional interaction between Vprand ANT.

[0044] A Plasma surface resonance sensorgrams of the interaction of ANTwith Vpr52-96, Vpr52-96[R73A, 80A] or an irrelevant control (Co). Onlythe sensorgram of the interaction with Vpr52-96 exhibits an increase ofbinding as a function of time and a positive signal at the start of thedissociation phase (off). The calculated K_(D) (K_(D)=k_(on)/k_(off)) ofthe interaction is 9.7±6.4 nM (X±SD, n=5).

[0045] B. Langmuir isotherm determined at different concentrations ofANT on sensorgrams corrected by substraction of the blank (sensorgramsobtained with Vpr52-96[R73A, 80A]).

[0046] C. Modulation of the Vpr52-96-ANT interaction by ANT ligands andANT-derived peptides. Measurements were performed as in A, in theabsence (Ø) or presence of bongkrekic acid (BA, 250 μM), atractyloside(Atr, 50 μM), the ANT104-116 peptide, or three control peptides (all at5 μM). ANT-2-derived peptide ANT104-116 [DKRTQFWRYFAGN] and controlpeptides (Co. I: scrambled ANT104-116 [FQNYWGHKRFRDA]; Co. II: mutatedANT104-116 [DGHKQFWGYFAGN]; Co. III: topologically equivalent peptide(aa 149-161) from the ANT-related human phosphate carrier protein[SNMLGEENTYLWR]. Activation or inhibition was calculated as(1−k_(0a)/k₀)×100, in which k_(0a) and k₀ are the initial velocity inthe presence or absence of the agent, respectively.

[0047] D. Langmuir isotherm for the binding of ANT104-116 tobiotinylated Vpr52-96 (as determined in A). The calculated K_(D) of theinteraction is 35 μM.

[0048] E. Schematic diagram showing the topology of ANT and the sequenceof the ANT-2-derived peptide ANT104-116.

[0049]FIG. 2 presents physical (A, B) and functional (C) interactionbetween Vpr and liposomes containing ANT.

[0050] A. Dose-response curve of FITC-labeled Vpr52-96 binding ontoANT-liposomes and plain liposomes.

[0051] B. Binding of FITC-Vpr52-96 (2 μM) to plain liposomes,ANT-proteoliposomes, in the presence or absence of BA (50 μM).

[0052] C. Permeabilization of ANT proteoliposomes by Vpr (X±SD, n=3).Liposomes were loaded with 4-methylumbelliferylphosphate (4-MUP) andexposed for 60 min to Atr (200 μM) or the indicated Vpr-derived peptides(1 μM), in the presence or absence of BA (50 μM), ADP (800 μM), and/orthe indicated peptides (same as in B, 0.5 μM, pre-incubated withVpr52-96 for 5 min). Then, alkaline phosphatase was added to convertliposome-released 4-MUP into the fluorochrome 4-methylumbelliferone(4-MU) and the percentage of 4-MUP release induced by Vpr-derivedpeptides was calculated as described in Material and Methods”.

[0053]FIG. 3 presents electrophysiological properties of Vpr52-96 andANT in planar lipid bilayers. Current fluctuations of Vpr52-96 (80 nM,+150 mV), Vpr52-96(0.4 nM, +100 mV), ANT (1 nM, +110 mV) andVpr52-96+ANT (0.4:1 nM, +115 mV) and associated histograms (right) ofconductance levels are shown.

[0054] A. Cooperative effect between ANT and Vpr52-96 at the singlechannel level. Current fluctuations of Vpr52-96 (80 nM, +150 mV),Vpr52-96 (0.4 nM, +100 mV), ANT (1 nM, +110 mV) and Vpr52-96+ANT (0.4:1nM, 115 mV) after incorporation into synthetic membranes. Single channelrecordings were performed using the “Tip-Dip” technique. The recordingsshown are representative for at least three independent determinations.

[0055] B. Statistical analysis of conductances obtained in A. Resultswere expressed as current distributions at different voltages.Conductances (γ; in picosiemens, pS) are calculated by division ofcurrent by voltage.

[0056]FIG. 4 presents oxidative properties of purified mitochondriaexposed to Vpr.

[0057] A. Oxygen consumption curves after addition of the indicatedagents. Trace a: control mitochondria (no pretreatment). Trace b:mitochondria pretreated for 10 min with 1 μM Vpr52-96. Numbers along thetraces are nmol of O₂ consumed min⁻¹ mg³¹ ¹ protein.

[0058] B. Respiratory control (RC) values calculated by dividing oxygenconsumption in the presence of CCCP by that measured with oligomycin(determined as in A), 10 min after addition of 1 μM of Vpr-derivedpeptides (mean values of 3 determinations).

[0059]FIG. 5 presents inner versus outer mitochondrial membranepermeabilization.

[0060] A. Respirometry performed after addition of NADH and Vpr52-96 (1μM). Numbers along the traces are nmol of O₂ consumed min⁻¹ mg⁻¹protein. Note that the Vpr-stimulated, NADH-dependent O₂ consumption wasfully sensitive to rotenone.

[0061] B. Kinetics of Vpr52-96 induced inner membrane permeabilizationto NADH and outer membrane permeabilization to reduced cytochrome c.Oxygen consumption was determined in the presence of 2 mM NADH (fullsquares) as in A (traces C-D) and cytochrome c (15 μM) oxidation (opencircles) was spectrofluorometrically measured, as described Rustin etal., 1994). The 100% value of cytochrome c oxidation was determined byaddition of 2.5 mM laurylmaltoside.

[0062] C. Kinetics of Vpr52-96-induced ΔΨ_(m) loss and cytochrome crelease. Purified mitochondria were treated with 1 μM Vpr52-96 subjectedto cytofluorometric determination of the percentage of mitochondriahaving a low ΔΨ_(m) using the ΔΨ_(m)-sensitive fluorochrome JC-1. Inparallel, cytochrome c was immunodetected in the supernatant ofmitochondria.

[0063]FIG. 6 presents Bcl-2-mediated inhibition of Vpr effects onmitochondria.

[0064] A. Vpr52-96-induced ΔΨ_(m) dissipation induced in intact cells.COS cells were microinjected with recombinant human Bcl-2 (10 μM),Königs polyanion (PA10, 2 μM), or PBS only, then incubated in theabsence (Co.) or presence of 1 μM Vpr52-96 for 3 hours, and stained withthe ΔΨ_(m)-sensitive dye JC-1 (2 μM; red fluorescence of mitochondriawith a high ΔΨ_(m), green fuorescence of mitochondria with a lowΔΨ_(m)).

[0065] B. Effect of Bcl-2 on the Vpr-induced inner MMP to NADH.Mitochondria were left untreated (Co.) or pretreated (10 min) with Bcl-2(0.8 μM) or BA (10 μM). Oxygen consumption of purified mitochondria wasmeasured as in FIG. 5 after addition of succinate+CCCP or NADH, asindicated.

[0066] C. Ultrastructural effects of Vpr on isolated mitochondria.Electron micrographs were obtained after incubation of mitochondria for5 or 15 min with 3 μM Vpr52-96, after pre-incubation (5 min) wuh 0.8 μMBcl-2 or 2 μM PA10.

[0067] D. Effect of Bcl-2 and PA-10 on Vpr52induced ΔΨ_(m) dissipationin purified mitochondria. Isolated mitochondria (200 μg protein per ml)were pre-incubated with the indicated inhibitors (5 μM CsA, 50 μM BA,0.8 μM Bcl-2, 2 μM PA10; 5-10 min), washed (10 min, 6800 g, 20° C.),incubated with the ΔΨ_(m)-sensitive dye JC-1 (200 nM, 10 min), exposedto Vpr52-96 (3 μM, 5 min), and subjected to flow cytometricdetermination of the fluorescence (570-595 nm) and the particle size(FSC). Numbers indicate the percentage of JC-1^(high) and JC-1^(low)mitochondria among ˜10⁴ events.

[0068] E. Quantitation of the frequency of JC-1^(low) mitochondria(X±SD, n=5) after incubation with different Vpr-derived peptides.Purified mitochondria were preincubated 10 min with or without Bcl-2(0.8 μM), Bcl-2Δα_(5/6) (0.8 μM) or BA (10 μM) in PT buffer, incubatedwith the ΔΨ_(m)-sensitive dye JC-1 (200 nM, 10 min), and then treated 5min with 3 μM of Vpr52-96 (wt, biotinylated, or modified as indicated),or 10 min. with 5-10 μM of Vpr-1-96, Vpr1-51, Vpr71-96, Vpr71-82 (wt ormodified as indicated), and finally subjected to flow cytometricanalysis as in D.

[0069]FIG. 7 presents differential effect of Bcl-2 and PA-10 on Vpr52-96binding to mitochondria.

[0070] A. Vpr52-96 binds mitochondria before inducing ΔΨ_(m) loss.Mitochondria were left unstained (insert in Co.) or exposed to theΔΨ_(m)-insensitive mitochondrial dye MitoTracker® Green (75 nM), alone(MTG) or with 0.5 μM of FITC-Vpr52-96; green fluorescence), incombination with the ΔΨ_(m)-sensitive mitochondrial dye MitoTracker® Red(CMXRos; red Fluorescence) followed by cytofluorometric two-coloranalysis. Numbers indicate the percentage of mitochondria in eachquadrant.

[0071] B. PA-10 but not Bcl-2 inhibit Vpr52-96 binding to mitochondria.Mitochondria were pre-incubated 10 min. with the indicated inhibitorsand the percentage of FITC-Vpr52-96-labeled mitochondria is determinedas in A. C. inhibitory effect of Bcl-2 on affinity purification of ANTby biotinylated Vpr52-96. Mitochondria were incubated with the indicatedinhibitors, and then exposed for 30 min at RT with 5 μM biotinylatedVpr52-96. Mitochondria were lysed either after incubation withbiotinylated Vpr52-96 (upper panel) or lysed before (lower panel) withTris/HCl as described in materials and methods. Biotinylated Vpr52-96complexed with its mitochondrial ligands was retained on avidin-agaroseand subjected to immunoblot detection of ANT.

[0072]FIG. 8 presents Bcl-2-mediated inhibition of the Vpr-ANTinteraction.

[0073] A. Plasmon surface resonance determination of the Bcl-2-mediatedinhibition of interaction between Vpr52-96 and native purified ANT. Theinteraction was measured after addition of the indicated concentrationsof recombinant Bcl-2, Bcl-2Δα_(5/6), or recombinant Bid, and data (X±SD,n=3) were calculated as in FIG. 1.

[0074] B. Effect of Bcl-2 on Vpr binding to ANT proteoliposomes. Theretention (X±SD, n=3) of FITC-labeled Vpr52-96 on ANT proteoliposomespreincubated with 800 nM of Bcl-2 or 2 μM PA10 was assessed as in FIG.2A.

[0075] C. Effect of Bcl-2 on the formation of Vpr-ANT channels in planarlipid bilayers. Single channel recordings (+75 mV) of Vpr52-96+ANT+Bax(0.4:1:0.3 nM) and Vpr52-96+ANT+Bcl-2 (0.4:1:1 nM) and correspondingamplitude histograms are displayed. Control values for Vpr52-96+ANTalone are similar as in FIG. 1e (not shown). c, closed; o, open.

[0076]FIG. 9 presents a model of the Vpr/PTPC interactions. Vpr crossesthe outer membrane through VDAC, which is inhibited by Koenig'spolyanion. Vpr then interacts with ANT. Bcl-2 and the ANT ligandbongkrekate inhibit the binding of Vpr to ANT, whereas CsA indirectlyaffects the pore forming function of ANT via its effect on cyclophilin D(Cyp-D).

DESCRIPTION OF THE EMBODIMENTS

[0077] The invention relates to discovery that the proapoptoticHIV-1-encoded protein Vpr induces mitochondrial membranepermeabilization via its physical and functional interaction with themitochondrial inner membrane protein ANT (adenine nucleotidetranslocator, also called ADP/ATP carrier). This is shown using avariety of different techniques: surface plasmon resonance,electrophysiology, synthetic proteoliposomes, studies on purifiedmitochondria (respirometry, electron microscopy, organellofluometry), aswell as microinjection of intact cells. The mode of action of Bcl-2 actson ANT and to prevent Vpr-mediated mitochondrial effects.

[0078] This invention relates to the discovery that Vpr primarilyaffects IM and not OM permeability in vitro. Vpr binds ANT in an ANTconformation-dependent fashion (FIGS. 1 and 2) and cooperates with ANTto form channels (FIG. 3) which permeabilize IM before OM becomespermeable to cytochrome c (FIGS. 4 and 5). Bcl-2 antagonizes thiseffect, based on two independent observations. First, its mode of actionclearly differs from that of the VDAC inhibitor PA10 (FIGS. 6 and 7).Second, Bcl-2 can affect the physical and functional ANT-Vpr interactionin a synthetic, VDAC-free system (FIG. 8). Although these data do notexclude the possibility that Bcl-2 and other members of Bcl-1 familymodulate the permeability of VDAC to relatively large, globular proteins(14.5 kDa for cytochrome c, as opposed to the linear, mostly α helicalstructure of Vpr52-96 resolved by NMR, W. Schüler, et al., J. Mol. Biol.285, 2105-2117 (1999)), they indicate that, at least in this particularmodel, Bcl-2 exerts its membrane-protective mitochondrial effect viaANT.

[0079] HIV-1 Viral protein R (Vpr) interacts with the permeabilitytransition pore complex (PTPC) to trigger mitochondrial membranepermeabilizaton (MMP) and consequent apoptosis. Vpr binds to the adeninenucleotide translocator (ANT), an inner mitochondrial membrane protein.E. Jacotot, et al., J. Exp. Med. 191, 33-45 (2000). When Vpr binds toANT, it cooperatively forms large conductance channels in syntheticmembranes. When added to purified mitochondria, Vpr uncouples therespiratory chain and induces a rapid inner MMP which precedes outer MMPto cytochrome c, Vpr-induced matrix swelling and inner MMP to protonsand NADH are prevented by preincubation of purified mitochondria withrecombinant Bcl-2 protein. In contrast to König's polyanion, a specificinhibitor of the voltage-dependent anion channel (VDAC), Bcl-2 fails toprevent Vpr from crossing the outer mitochondrial membrane. Bcl-2reduces the ANT-Vpr interaction and abolishes channel formation by theANT-Vpr complex. Hence, both Vpr and Bcl-2 modulate MMP through a directinteraction with ANT.

[0080] Methods of Altering or Preventing Binding of Vpr to ANT

[0081] The discovery of the physical and functional interaction of Vprwith ANT enables methods of altering or preventing binding of Vpr toANT. As illustrated in Examples 1-4, the interaction of Vpr to ANT canbe detected and modulated in a variety of different assay systems. Forexample, Bcl-2 modulates the physical and functional interaction of Vprwith ANT. Likewise, a peptide, ANT104-116, corresponding to the overlapbetween the Bcl-2 binding motif and the second ANT loop inhibits ANT-Vprbinding. Thus, these molecules can be used to alter or prevent bindingof Vpr to ANT. Other peptidic or non-peptidic molecules can be designedto similarly inhibit this binding.

[0082] The identification of Vpr-ANT binding allows the generation ofmolecules that can modulate apoptosis. The methods presented in theExamples, and other conventional techniques, can be adapted to screenfor Vpr, Bcl-2, or ANT variants, or other polypeptides or molecules thataffect Vpr-ANT binding. This allows for the generation of moleculescapable of enhancing or inhibiting Vpr-ANT binding. The activity ofthese molecules can be assessed by competitive binding assays. Forexample, molecules can be assessed for there ability to inhibit ANT-Vprbinding using the binding assays described in the Examples. The, skilledartisan understands that many other techniques could similarly be used.The identified molecules can be further assessed for apoptotic activityby conventional techniques. Furthermore, based on the structure of Vprmolecules determined to bind to ANT, structurally similar molecules canbe designed to mimic Vpr activity or to inhibit this activity.

[0083] In one embodiment, soluble versions of Vpr or ANT polypeptidescan be incubated with cells to enhance or inhibit the induction ofapoptosis. In one embodiment these polypeptides contain mutations thatinterfere with apoptosis. In another embodiment, these polypeptidescontain mutations that enhance apoptosis. In one embodiment, thesepolypeptides are synthetic. In another embodiment, these polypeptidesare produced by recombinant techniques.

[0084] Vpr and ANT polypeptides and peptides of greater than 9 aminoacids that inhibit or augment Vpr-ANT binding, mitochondrial membranepermeabilization, or apoptosis are an embodiment of the invention, aswell as peptides that are at least 10-20, 20-30, 30-50, 50-100, and100-365 amino acids in size. DNA fragments encoding these polypeptidesand peptides are encompassed by the invention.

[0085] Synthetic polypeptides and peptides can be generated by a varietyof conventional techniques. Such techniques include those described inB. Merrifield, Methods Enzymol. 289:3-13, 1997, H. Ball and P. Mascagni,Int. J. Pept. Protein Res. 48:31-47, 1996; F. Molina et al., Pept. Res.9:151-155, 1996; J. Fox, Mol. Biotechnol. 3:249-258, 1995; and P. Lepageet al., Anal. Biochem. 213: 40-48, 1993.

[0086] In another embodiment, peptides can be prepared by subcloning aDNA sequence encoding a desired peptide sequence into an expressionvector for the production of the desired peptide. The DNA sequenceencoding the peptide is advantageously fused to a sequence encoding asuitable leader or signal peptide. Alternatively, the DNA fragment maybe chemically synthesized using conventional techniques. The DNAfragment can also be produced by restriction endonuclease digestion of adone of, for example HIV-1, DNA using known restriction enzymes (NewEngland Biolabs 1997 Catalog, Stratagene 1997 Catalog, Promega 1997Catalog) and isolated by conventional means, such as by agarose gelelectrophoresis.

[0087] In another embodiment, the well known polymerase chain reaction(PCR) procedure can be employed to isolate and amplify a DNA sequenceencoding the desired protein fragment. Oligonucleotides that define thedesired termini of the DNA fragment are employed as 5′ and 3′ primers.The oligonucleotides can contain recognition sites for restrictionendonucleases, to facilitate insertion of the amplified DNA fragmentsinto an expression vector. PCR techniques are described in Saiki et al.,Science 239:487 (1988); Recombinant DNA Mythology, Wu et al., eds.,Academic Press, Inc., San Diego (1989), pp. 189-196: and PCR Protocols:A Guide to Methods and Applications, Innis et al., ed., Academic Press,Inc., (1990). It is understood of course that many techniques could beused to prepare polypeptide and DNA fragments, and that this embodimentin no way limits the scope of the invention.

[0088] Screening Methods with Vpr and ANT

[0089] Vpr or ANT polypeptides can be assessed for their ability tomediate apoptosis, as well as to block Vpr mediated apoptosis. Forexample, fragments of Vpr can be assessed for their ability to blocknative Vpr binding to ANT by conventional titration experiments.

[0090] In one embodiment, surface plasmon resonance is used to assessbinding of Vpr to ANT as described herein. In another embodiment,electrophysiology is used to assess binding of Vpr to ANT as describedherein. In another embodiment, purified mitochondria are used to assessbinding of Vpr to ANT as described herein. In another embodiment,synthetic proteoliposomes are used to assess binding of Vpr to ANT asdescribed herein. In another embodiment, microinjection of live cells isused to assess binding of Vpr to ANT as described herein. It isunderstood of course that many techniques could be used to assessbinding of Vpr to ANT, and that these embodiments in no way limit thescope of the invention.

[0091] In another embodiment the yeast two-hybrid system developed atSUNY (described in U.S. Pat. No. 5,283,173 to Fields et al.; J. Lubanand S. Goff., Curr Opin. Biotechnol. 6:59-64, 1995; R. Brachmann and J.Boeke, Curr Opin. Biotechnol. 8:561-568, 1997; R. Brent and R. Finley,Ann. Rev. Genet. 31:663-704, 1997; P. Bartel and S. Fields, MethodsEnzymol. 254:241-263, 1995) can be used to screen for a inhibitors ofthe Vpr-ANT interaction as follows. Vpr, or portions thereof responsiblefor interaction, can be fused to the Gal4 DNA binding domain andintroduced, together with an ANT molecule fused to the Gal 4transcriptional activation domain, into a strain that depends on Gal4activity for growth an plates lacking histidine. Interaction of the Vprpolypeptide with an ANT molecule allows growth of the yeast containingboth molecules and allows screening for the molecules that inhibit oralter this interaction (i.e., by inhibiting or augmenting growth).

[0092] In an alternative embodiment a detectable marker (e.g.β-galactosidase) can be used to measure binding in a yeast hybrid assay.

[0093] In addition, the identification of Vpr as an ANT-binding moleculeallows methods of detecting and quantifying ANT expression in cells. Forexample, by contacting a labeled Vpr polypeptide with a biologicalsample comprising ANT and detecting the Vpr-ANT complex, the level ofANT can be determined.

[0094] Purified ANT polypeptides (including proteins, polypeptides,fragments, variants, oligomers, and other forms) may be tested for theability to bind Vpr in any suitable assay, such as a conventionalbinding assay. Similarly, Vpr polypeptides (including proteins,polypeptides, fragments, variants, oligomers, and other forms) may betested for the ability to bind ANT. To illustrate, the Vpr polypeptidemay be labeled with a detectable reagent (e.g., a radionucleotide,chromophore, enzyme that catalyzes a colorimetric or fluorometricreaction, and the like). The labeled polypeptide is contacted with cellsexpressing ANT. The cells then are washed to remove unbound labeledpolypeptide, and the presence of cell-bound label is determined by asuitable technique, chosen according to the nature of the label.

[0095] Alternatively, the binding properties of Vpr polypeptides andpolypeptide fragments can be determined by analyzing the binding of Vprpolypeptides and polypeptide fragments to ANT-expressing cells by FACSanalysis and/or immunofluorescence. This allows the characterization ofthe binding of Vpr and ANT polypeptides and polypeptide fragments, andthe discrimination of relative abilities of Vpr polypeptides andpolypeptide fragments to bind to ANT. In vitro binding assays with Vprand ANT can similarly be used to characterize Vpr-ANT binding activity.

[0096] Another type of suitable binding assay is a competitive bindingassay. To illustrate, biological activity of a variant may be determinedby assaying for the variant's ability to compete with the native proteinfor binding to Vpr or ANT.

[0097] Competitive binding assays can be performed by conventionalmethodology. Reagents that may be employed in competitive binding assaysinclude radiolabeled Vpr and intact cells expressing ANT (endogenous orrecombinant). For example, a radiolabeled Vpr fragment can be used tocompete with a soluble Vpr variant for binding to ANT in cells, insteadof intact cells, one could substitute ANT protein bound to a solidphase.

[0098] Another type of competitive binding assay utilizes radiolabeledVpr and isolated mitochondria. Qualitative results can be obtained bycompetitive autoradiographic plate binding assays, while Scatchard plots(Scatchard, Ann. N.Y. Acad. Sci. 51:660, 1949) may be utilized togenerate quantitative results.

[0099] Peptidic or Non-Peptidic Molecules that Affect Interaction of Vprto ANT or Mimic Its Capacity to Interact with ANT

[0100] Variants

[0101] The invention encompasses variants of Vpr or ANT that are alteredin their binding activity. Among the variant polypeptides providedherein are variants of native polypeptides that retain the nativebiological activity or the substantial equivalent thereof. One exampleis a variant that binds with essentially the same binding affinity asdoes the native form. Binding affinity can be measured by conventionalprocedures, e.g., as described in U.S. Pat. No. 5,512,457 and as setforth below.

[0102] Variants may also bind with increased affinity. In oneembodiment, a variant is an agonist of the native Vpr for ANT'sbiological activity. In another embodiment, a variant is an antagonistof the native Vpr for ANT's biological activity. Agonistic orantagonistic activity can be readily determined by the proceduresdescribed herein.

[0103] Variants include polypeptides that are substantially homologousto the native form, but which have an amino acid sequence different fromthat of the native form because of one or more deletions, insertions orsubstitutions. Particular embodiments include, but are not limited to,polypeptides that comprise from one to ten deletions, insertions orsubstitutions of amino acid residues, when compared to a nativesequence.

[0104] A given amino acid may be replaced, for example, by a residuehaving similar physiochemical characteristics. Examples of suchconservative substitutions include substitution of one aliphatic residuefor another, such as Ile, Val, Leu, or Ala for one another,substitutions of one polar residue for another, such as between Lys andArg, Glu and Asp, or Gln and Asn; or substitutions of one aromaticresidue for another, such as Phe, Trp, or Tyr for one another. Otherconservative substitutions, e.g., involving substitutions of entireregions having similar hydrophobicity characteristics, are well known.Variants can be generated using conventional techniques including randomor site-directed mutagenesis.

[0105] Antibodies

[0106] Within an aspect of the invention, Vpr and ANT polypeptides, andpeptides based on the amino acid sequence of Vpr and ANT, can beutilized to prepare antibodies that specifically bind to Vpr and ANTpolypeptides. Antibodies that are immunoreactive with the polypeptidesof the invention are provided herein. In this aspect of the invention,the polypeptides based on the amino acid sequence of Vpr and ANT can beutilized to prepare antibodies that specifically bind to Vpr and ANT.Such antibodies specifically bind to the polypeptides via theantigen-binding sites of the antibody (as opposed to non-specificbinding). Thus, the polypeptides, fragments, variants, fusion proteins,etc., as set forth above may be employed as immunogens in producingantibodies immunoreactive therewith. More specifically, thepolypeptides, fragment, variants, fusion proteins, etc. containantigenic determinants or epitopes that elicit the formation ofantibodies.

[0107] These antigenic determinants or epitopes can be either linear orconformational (discontinuous). Linear epitopes are composed of a singlesection of amino acids of the polypeptide, while conformational ordiscontinuous epitopes are composed of amino acids sections fromdifferent regions of the polypeptide chain that are brought into closeproximity upon protein folding (C. A. Janeway, Jr. and P. Travers,Immuno Biology 3:9 (Garland Publishing Inc., 2nd ed. 1996)). Becausefolded proteins have complex surfaces, the number of epitopes availableis quite numerous; however, due to the conformation of the protein andsteric hinderances, the number of antibodies that actually bind to theepitopes is less than the number of available epiopes (C. A Janeway, Jr.and P. Travers, Immuno Biology 2:14 (Garland Publishing Inc., 2nd ed.1996)). Epitopes may be identified by any of the methods known in theart.

[0108] Thus, one aspect of the present invention relates to theantigenic epitopes of the polypeptides of the invention. Such epitopesare useful for raising antibodies, in particular monoclonal antibodies,as described in detail below. Additionally, epitopes from thepolypeptides of the invention can be used as research reagents, inassays, and to purify specific binding antibodies from substances suchas polyclonal sera or supernatants from cultured hybridomas. Suchepitopes or variants thereof can be produced using techniques well knownin the art such as solid-phase synthesis, chemical or enzymatic cleavageof a polypeptide, or using recombinant DNA technology.

[0109] As to the antibodies that can be elicited by the epitopes of thepolypeptides of the invention, whether the epitopes have been isolatedor remain part of the polypeptides, both polyclonal and monoclonalantibodies may be prepared by conventional techniques as describedbelow.

[0110] The term “antibodies” is meant to include polyclonal antibodies,monoclonal antibodies, fragments thereof, particular antigen bindingfragments such as F(ab′)2 and Fab fragments, as well as anyrecombinantly produced binding partners. Antibodies are defined to bespecifically binding if they bind with a K_(a) of greater than or equalto about 10⁷ M⁻¹. Affinities of binding partners or antibodies can bereadily determined using conventional techniques, for example thosedescribed by Scatchard et al., Ann. N.Y Acad. Sci., 51:660 (1949).

[0111] Polyclonal antibodies can be readily generated from a variety ofsources, for example, horses, cows, goats, sheep, dogs, chickens,rabbits, mice, or rats, using procedures that are well known in the art.In general, purified Vpr (or ANT) or a peptide based on the amino acidsequence of Vpr (or ANT) that is appropriately conjugated isadministered to the host animal typically through parenteral injection.The immunogenicity of Vpr (or ANT) can be enhanced through the use of anadjuvant, for example, Freund's complete or incomplete adjuvant.Following booster immunizations, small samples of serum are collectedand tested for reactivity to Vpr or ANT polypeptide. Examples of variousassays useful for such determination include those described inAntibodies: A Laboratory Manual, Harlow and Lane (eds.) Cold SpringHarbor Laboratory Press, 1988; as well as procedures, such ascountercurrent immuno-electrophoresis (CIEP), radioimmunoassay,radio-immunoprecipitation, enzyme-linked immunosorbent assays (ELISA),dot blot assays, and sandwich assays. See U.S. Pat. Nos. 4,376,110 and4,486,530.

[0112] Monoclonal antibodies can be readily prepared using well knownprocedures. See, for example, the procedures described in U.S. Pat. Nos.RE 32,011, 4,902,614, 4,543,439, and 4,411,993; Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Plenum Press,Kennett, McKearn, and Bechtol (eds.). 1980. Briefly, the host animals,such as mice, are injected intraperitoneally at least once andpreferably at least twice at about 3 week intervals with isolated andpurified Vpr (or ANT), conjugated Vpr (or ANT) peptide, optionally inthe presence of adjuvant. Mouse sera are then assayed by conventionaldot blot technique or antibody capture (ABC) to determine which animalis best to fuse. Approximately two to three weeks later, the mice aregiven an intravenous boost of Vpr (or ANT) or conjugated Vpr (or ANT)peptide. Mice are later sacrificed and spleen cells fused withcommercially available myeloma cells, such as Ag8.653 (ATCC), followingestablished protocols. Briefly, the myeloma cells are washed severaltimes in media and fused to mouse spleen cells at a ratio of about threespleen cells to one myeloma cell. The fusing agent can be any suitableagent used in the art, for example, polyethylene glycol (PEG). Fusion isplated out into plates containing media that allows for the selectivegrowth of the fused cells. The fused cells can then be allowed to growfor approximately eight days. Supernatants from resultant hybridomas arecollected and added to a plate that is first coated with goat anti-mouseIg. Following washes, a label, such as ¹²⁵I-Vpr (or ¹²⁵I-ANT), is addedto each well followed by incubation. Positive wells can be subsequentlydetected by autoradiography. Positive clones can be grown in bulkculture and supernatants are subsequently purified over a Protein Acolumn (Pharmacia).

[0113] The monoclonal antibodies of the invention can be produced usingalternative techniques, such as those described by Alting-Mees et al.,“Monoclonal Antibody Expression Libraries; A Rapid Alternative toHybridomas”, Strategies in Molecular Biology 3:1-9 (1990), which isincorporated herein by reference. Similarly, binding partners can beconstructed using recombinant DNA techniques to incorporate the variableregions of a gene that encodes a specific binding antibody. Such atechnique is described in Larrick et at., Biotechnology, 7:394 (1989).

[0114] The monoclonal antibodies of the present invention includechimeric antibodies, e.g., humanized versions of murine monoclonalantibodies. Such humanized antibodies may be prepared by knowntechniques, and offer the advantage of reduced immunogenicity when theantibodies are administered to humans. In one embodiment, a humanizedmonoclonal antibody comprises the variable region of a murine antibody(or just the antigen binding site thereof) and a constant region derivedfrom a human antibody. Alternatively, a humanized antibody fragment maycomprise the antigen binding site of a murine monoclonal antibody and avariable region fragment (lacking the antigen-binding site) derived froma human antibody. Procedures for the production of chimeric and furtherengineered monoclonal antibodies include those described in Riechmann etal. (Nature 332:323, 1988), Liu et al. (PNAS 84:34-39, 1987), Larrick etal. (Bio/Technology 7:934, 1989), and Winter and Harris (TIPS 14:139,May, 1993). Procedures to generate antibodies transgenically can befound in GB 2,272,440, U.S. Pat. Nos. 5,569,825 and 5,545,806 andrelated patents claiming priority therefrom, all of which areincorporated by reference herein.

[0115] Once isolated and purified, the antibodies against Vpr and ANT,and other Vpr and ANT binding proteins, can be used to detect thepresence of Vpr and ANT in a sample using established assay protocols.Further the antibodies of the invention can be used therapeutically tobind to Vpr or ANT and inhibit its activity in vivo.

[0116] Antibodies directed against Vpr or ANT and other Vpr or ANTbinding proteins can be used to modulate the biological activity of Vprand ANT. One class of these antibodies produce mitochondrial membranepermeabilization and apoptosis. In contrast, another class of theseantibodies can inhibit mitochondrial membrane permeabilization andapoptosis.

[0117] Those antibodies that additionally can block Vpr-ANT binding ofmay be used to inhibit a biological act that results from such binding.Such blocking antibodies may be identified using any suitable assayprocedure, such as by testing antibodies for the ability to inhibitbinding of Vpr to ANT. Alternatively, blocking antibodies may beidentified in assays for the ability to inhibit a biological effect thatresults from binding of Vpr to ANT in cells.

[0118] Such an antibody may be employed in an in vitro procedure, oradministered in vivo to inhibit a biological activity mediated by theentity that generated the antibody. Disorders caused or exacerbated(directly or indirectly) by the interaction of Vrp with ANT. Atherapeutic method involves in vivo administration of a blockingantibody to a mammal in an amount effective in inhibiting ANT-mediatedapoptosis. Monoclonal antibodies are generally preferred for use in suchtherapeutic methods. In one embodiment, an antigen-binding antibodyfragment is employed.

[0119] Antibodies may be screened for agonistic (i.e., ligand-mimicking)properties. Such antibodies, upon binding to ANT, induce biologicaleffects (e.g., apoptosis) similar to the biological effects induced whenVpr binds to ANT. Agonistic antibodies may be used to induceANT-mediated apoptosis of cells.

[0120] Compositions comprising an antibody that is directed against Vpror ANT, and a physiologically acceptable diluent, excipient, or carrier,are provided herein. Suitable components of such compositions are asdescribed above for compositions containing Vpr or ANT.

[0121] Also provided herein are conjugates comprising a detectable(e.g., diagnostic) or therapeutic agent, attached to the antibody.Examples of such agents are presented above. The conjugates find use inin vitro or in vivo procedures.

[0122] Other Molecules

[0123] The invention also encompasses molecules that compete for orenhance the binding of Vpr to ANT, which can be identified through thescreening assays described herein or by structure-based design using,for example, molecular modeling of Vpr-ANT binding.

[0124] Pharmaceutical and Diagnostic Compositions

[0125] Compositions of the present invention may contain a peptidic ornon-peptidic molecules in any form, such as native proteins, variants,derivatives, oligomers, and biologically active fragments. In particularembodiments, the composition comprises a soluble polypeptide or anoligomer comprising soluble Vpr, Bcl-2, or ANT polypeptides orfragments.

[0126] Compositions comprising an effective amount of a molecule of thepresent invention, in combination with other components such as aphysiologically acceptable diluent carrier, or excipient, are providedherein. The molecules can be formulated according to known methods usedto prepare pharmaceutically useful compositions. They can be combined inadmixture, either as the sole active material or with other known activematerials suitable for a given indication, with pharmaceuticallyacceptable diluents (e.g., saline, Tris-HCl, acetate, and phosphatebuffered solutions), preservatives (e.g., thimerosal, benzyl alcohol,parabens), emulsifiers, solubilizers, adjuvants and/or carriers.Suitable formulations for pharmaceutical compositions include thosedescribed in Remington's Pharmaceutical Sciences, 16th ed. 1980, MackPublishing Company, Easton, Pa.

[0127] In addition, such compositions can be complexed with polyethyleneglycol (PEG), meal ions, or incorporated into polymeric compounds suchas polyacetic acid, polyglycolic acid, hydrogels, dextran, etc., orincorporated into liposomes, microemulsions, micelles, unilamellar ormultilamellar vesicles, erythrocyte ghosts or spheroblasts. Suchcompositions will influence the physical state, solubility, stability,rate of in vivo release, and rate of in vivo clearance, and are thuschosen according to the intended application.

[0128] The compositions of the invention can be administered in anysuitable manner, e.g., topically, parenterally, or by inhalation. Theterm “parenteral” includes injection, e.g., by subcutaneous,intravenous, or intramuscular routes, also including localizedadministration, e.g., at a site of disease or injury. Sustained releasefrom implants is also contemplated. One skilled in the pertinent artwill recognize that suitable dosages will vary, depending upon suchfactors as the nature of the disorder to be treated, the patient's bodyweight, age, and general condition, and the route of administration.Preliminary doses can be determined according to animal tests, and thescaling of dosages for human administration is performed according toart-accepted practices.

[0129] Compositions comprising nucleic acids in physiologicallyacceptable formulations are also contemplated. DNA may be formulated forinjection, for example.

[0130] Methods for Screening for ANT Alterations

[0131] The invention also provides methods for screening for genetic orepigenetic alterations in the expression or structure of the three ANTisoforms in humans.

[0132] The invention provides for diagnosis of diseases associated withaberrant ANT expression. For example, a particular cancer may have aspecific modification of ANT associated with it. Diagnosis of thatcancer can be achieved by using Vpr or a fragment of Vpr capable ofretaining binding to ANT in a binding assay, for example, as describedherein. The expression or structure of the three ANT isoforms inpatients can thereby be determined and a diagnosis achieved.

[0133] Methods for Specific Cell Killing

[0134] Vpr, or a biologically active fragment thereof such as vpr52-96,can be used to induce apoptosis in cells. In one embodiment, a vpr52-96peptide is fused to molecule for targeting to a specific cell type andinduces apoptosis in that cell type. In a further embodiment, aVpr-targeting molecule conjugate specifically kills cancer cells. Themethodology can be similar to the successful use of a recombinantchimeric protein containing interleukin 2 (IL-2) protein fused to Bax toselectively kill IL-2 receptor-bearing cells in vitro R. Aqeilan, S.Yarkoni, and H. Lorberbourn-Galski, FEBS Lett 457:271-6 (1999).

[0135] In other embodiments, biologically active Vpr-targeting moleculeconjugates can be used to specifically target and kill other cell typesinvolved in disease.

[0136] Double Test

[0137] To study ANT's role in apoptosis and, more specifically, ANT'srole in the permeabilization of mitochondrial membranes (Brenner et al.,Oncogene, 2000; Costantini at al., Oncogene, 2000), the inventors havedeveloped a functional double test that makes it possible to measuresimultaneously the antiport (vital) function and the pore (lethal)function of ANT in artificial lipid double-layers or liposomes.

[0138] The principle of the functional double test is based on thereconstitution of ANT in liposomes, the encapsulation of differentsubstrates (fluorescent substrate and ATP) in the interior ofproteoliposomes, the addition of enzymes and ADP to the exterior of theliposomes, and then measurement by fluorescence of the salting out of asubstrate through the pore formed by ANT and, at the same time, themeasurement by luminescence of ATP translocated in response to erogenousADP. Any peptide or non-peptide compound can be reconstituted with ANTduring the formation of liposomes (e.g., Bax, Bcl-2, Bcl-x(L)),encapsulated in liposomes or added in an external manner toproteoliposomes (e.g., addition of peptide molecules [e.g., Vpr,Vpr52-96], Bid, etc.) or not (atractyloside, calcium, t-BHP, diamide,BA. cyclosporine A., verteporfin, etc.) to determine its impact on thetwo functions of ANT. Quantitative measurements can be performed in afixed point or kinetic manner. This test is operation in 96 wellmicroplates and can be adapted to HTS (high throughput screening).

[0139] This functional double test enables the screening of moleculesthat induce or inhibit apoptosis, of ANT partner molecules capable oftransforming or of facilitating the ANT-to-pore transformation, or thediagnosis of particular functional forms of ANT (alteration of vitaland/or lethal functions, alterations of the ratio of ANT isoforms). Theantiport function test makes it possible to evaluate the toxicity ofmolecules vis-à-vis the vital function of ANT.

[0140] The specification is most thoroughly understood in light of theteachings of the references cited within the specification which arehereby incorporated by reference. The embodiments within thespecification and the examples provide an illustration of embodiments ofthe invention and should not be construed to limit the scope of theinvention. The skilled artisan readily recognizes that many otherembodiments are encompassed by the invention.

EXAMPLE 1

[0141] Physical and Functional Interaction between Vpr and ANT

[0142] Surface plasmon resonance measurements indicate that purifieddetergent-solubilized ANT protein binds to the immobilized VprC-terminal moiety biotin-Vpr52-96 (but to a far lesser extent to mutatedbiotin-Vpr52-96[R73,80A]) with an affinity in the nanomolar range (FIGS.1A and B). This interaction is modulated by two ANT ligands whichdifferentially affect ANT conformation (M. Klingenberg, J. MembraneBiol. 56, 97-105 (1980)), namely the PTPC activator atractyloside (Atr).which favors Vpr binding, and the PTPC inhibitor bongkrekic acid (BA),which reduces Vpr binding (FIG. 1C). Vpr52-96 binding to membranes isgreatly facilitated in liposomes in which ANT has been reconstituted ascompared to protein-free liposomes (FIG. 2A). This ANT effect isinhibited by BA (FIG. 2B). Vpr52-96 (but not the N-terminal moiety ofVpr [Vpr1-51] nor mutated Vpr52-96. in which arginine 77 is replaced byalanine, Vpr52-96[R77A]) also causes permeabilization of ANTproteoliposomes (FIG. 2C), yet has no effect on plain liposomes.

[0143] Bcl-2-like proteins bind to a motif of ANT (aa 105-155), I.Marzo. et al., Science 281, 2027-2031 (1998), whose implication inapoptosis control has been confirmed by deletion mapping of ANT. M. K.A. Bauer. A Schubert O. Rocks, S. Grimm, J. Cell Biol. 147, 1493-1501(1999). This motif partially overlaps with the second ANT loop (aa92-116), a regulatory domain exposed to the intermembrane space. G.Brandolin, A Le-Saux, V. Trezeguet, G. J. Lauquinn, P. V. Vignais. J.Bioenerg. Biomembr. 25, 493-501 (1993). M. Klingenberg. J. Bioenerg.Biomembr. 25, 447-457 (1993). A peptide corresponding to the overlapbetween the Bcl-2 binding motif and this loop (ANT104-116) inhibited theANT Vpr interaction (FIG. 1C), presumably via direct association withVpr52-96 (insert in FIG. 1D).

[0144] Neither a topologically-related peptide motif derived from thehuman phosphate carrier nor mutated and scrambled versions of ANT104-116(control peptides in FIG. 1C) had such inhibitory effects. ANT104-116(but not the control peptides) also prevented Vpr52-96-induced membranepermeabilizion of ANT proteoliposomes (FIG. 2C), indicating that in thecontext of the lipid bilayer, me effect of Vpr involved a directinteraction with ANT. In planar lipid bilayers, low doses of Vpr52-96(<1 nM) were incapable of forming channels, unless ANT was present.

[0145] ANT and Vpr52-96 cooperated to form discrete channels whoseconductance (190±2 pS) (FIG. 3) was much larger than those formed byhigh doses (80 nM) of Vpr52-96 alone (55±2 pS) (FIG. 3 and S. C. Piller,G. D. Ewart, A. Prekumar, G. B. Cox, P. W. Gage, Proc. Natl. Acad. Sci.USA 93, 11-1-115 (1996)), yet was in the range of those formed byCa²⁺-treated ANT (N. Brustovetsky, M. Klingenberg, Biochemistry 35,8483-8488 (1996). C. Brenner. et al., Oncogene 19, 329-336 (2000). Thesebiophysical experiments demonstrate that ANT and Vpr directly interactin membranes to form functionally competent channel-forminghetero(poly)mers.

EXAMPLE 2

[0146] Oxidative Properties of Purified Mitochondria Exposed to Vpr.

[0147] As compared to untreated organelles (FIG. 4A trace a), purifiedmitochondria preincubated with Vpr52-96 (FIG. 4A, trace b) exhibited agross deficiency in respiratory control (RC). Vpr increased succinateoxidation preceding ADP addition and abolished both the inhibitoryeffect of oligomycin (a specific ATPase inhibitor) and the stimulatoryeffect of uncoupling by the protonophore carbamoyl cyanidem-chlorophenylhydrazone (CCCP). Thus, Vpr52-96 (but not Vpr1-51) reducedthe RC (ratio of oxygen consumption with oligomycin versus CCCP) to avalue of 1.1, as compared to 5.3 in control mitochondria (FIG. 4B). Theentire Vpr protein (Vpr1-96), and a short peptide corresponding to theminimum “mitochondriotoxic” motif of Vpr (Vpr71-82), (L G. Macreadie, etal., Proc. Natl. Acad. Sci. USA 92, 2770-2774 (1995). I. G. Macreadie,et al., FEBS Lett. 410, 145-149 (1997); E. Jacotot, et al., J. Exp. Med.191, 33-45 (2000)) also reduced the RC values (FIG. 4B) Noticeably, theVpr-induced loss of RC was not associated with a significant decrease ofthe oxidation rate (FIG. 4A), suggesting that no major loss ofmembrane-bound cytochrome c occurred upon short-term incubation withVpr52-96. Accordingly, adding cytochrome c to Vpr52-96-treatedmitochondria oxidizing succinate did not stimulate the rate of oxygenuptake (FIG. 4A, trace b). The observation of Vpr-mediated uncoupling ofthe respiratory chain prompted us to test its capacity to induce IMpermeabilization. The IM being essentially impermeable to NADH (P.Rustin, et al., J. Biol. Chem. 271, 14785-14790 (1996), no significantoxygen uptake could be measured when NADH was added to controlmitochondria (FIG. 5A, trace a). However, addition of Vpr52-96 prompteda significant, NADH-stimulated oxygen consumption (FIG. 5A, trace b).This indicates that Vpr permeabilized IM both to protons (leading touncoupling, FIG. 4A trace b) and to NADH (FIG. 5A, trace b).

[0148] The differential kinetics of inner and outer MMP to NADH andcytochrome c, respectively, were assessed FIG. 5B). NADH oxidation bymitochondria added with Vpr52-96 was found maximal after 10 min (FIG.5B). Under similar conditions, Vpr52-96 only induced a marginal accessof cytochrome c to cytochrome c oxidase (FIG. 5B). Moreover, the ΔΨ_(m)loss occurred well before cytochrome c release can be detected byimmunoblot (FIG. 5C). Hence, Vpr52-96 causes inner MMP well before OMbecomes permeable to exogenous cytochrome c. Accordingly, at theultrastructural level (see below. FIG. 6C), Vpr52-96 treatedmitochondria exhibited matrix swelling before OM rupture becameapparent.

EXAMPLE 3

[0149] Bcl-2-Mediated Inhibition of Vpr Effects on Mitochondria.

[0150] Extracellular addition of Vpr to intact cells induced a rapidΔΨ_(m) loss, before nuclear condensation occurred. These effects wereprevented by microinjection of recombinant Bcl-2 into the cytoplasm(FIG. 6A). Preincubation of purified mitochondria with recombinant Bcl-2(or the ANT ligand BA) also prevented the Vpr-mediated inner MMP to NADH(FIG. 6B). Concomitantly, both the Vpr-induced matrix swelling (FIG. 6C)and ΔΨ_(m) loss (FIGS. 6D and E) were inhibited by Bcl-2 (but not byBcl-2Δα5/6, a deletion mutant lacking the putative pore-forming α5 andα6 helices, S. Schendel, M. Montal, J. C. Reed, Cell Death Differ. 5,372-380 (1998)), by two pharmacological inhibitors of the PTPC (BA andcyclosporin A; CsA), as well as by the specific VDAC inhibitor Koenig'spolyanion (PA10; S. Stanley, J. A. Dias, D. D'Arcangelis, C. A.Mannella, J. Biol. Chem. 270, 16694-16700 (1995)). Microinjected PA10also inhibits the effect of Vpr52-96 on intact cells (FIG. 6A). Bindingof Vpr52-96 to purified mitochondria was completely abolished bypre-incubation of the organelles with PA10, partially reduced by BA, butnot affected by CsA (FIG. 7B). Thus, Vpr must access mitochondriathrough VDAC.

[0151] Bcl-2 may be expected to prevent Vpr from crossing OM via VDAC(based on the Bcl-2 mediated closure of VDAC) (S. Shimizu M. Narita, Y.Tsujimoto, Nature 399, 483-487 (1999). S. Shimizu, A. Konishi, T.Kodama, Y. Tsujimoto, Proc. Natl. Acad. Sci. USA 97, 3100-3105 (2000))and/or to inhibit the Vpr effect on ANT (based on its physical andfunctional interaction wit ANT) (I. Marzo, et al., Science 281,2027-2031 (1998); C. Brenner, et al., Oncogene 19, 329-336 (2000); M.Narita, et al., Proc., Natl. Acad. Sci. USA 95, 14681-14686 (1998)).Although recombinant Bcl-2 strongly reduced the Vpr52-96-induced matrixswelling (FIG. 6C) and ΔΨm loss (FIGS. 6D and E), it failed to impairthe binding of Vpr52-96 to purified mitochondria (FIG. 7B). Thedifferential inhibitory effects of PA10 and Bcl-2 on the Vpr-ANTinteraction was confirmed in a distinct experimental system. PA10 fullyabolished the affinity-mediated purification of ANT using biotinylatedVpr52-96 (FIG. 7C), provided that its effect was assessed on intactmitochondria (in which Vpr52-96 has to cross OM to reach ANT). Incontrast, PA10 did not affect the Vpr52-96-mediated purification of ANTfrom triton-solubilized mitochondria (in which ANT is readily accessibleto Vpr52-96). In the same conditions, Bcl-2 reduced theVpr52-96-mediated recovery of ANT, irrespective of its addition tointact or solubilized mitochondria (FIG. 7C). Thus, Bcl-2 does notinterfere with the (PA10 inhibited) VDAC-mediated conduit allowingVpr52-96 to pass OM.

EXAMPLE 4

[0152] Bcl-2Mediated Inhibition of the Vpr-ANT Interaction.

[0153] A further series of experiments indicated that Bcl-2 modulatedthe physical and functional interaction of Vpr with ANT. RecombinantBcl-2 (but not Bcl-2 Δα5/6) reduced Vpr52-96 binding to soluble (FIG.8A) or membrane-associated (FIG. 8B) ANT. Since Bcl-2 did not bindVpr52-96, inhibition of the Vpr/ANT binding is likely due to a directBcl-2/ANT interaction (I. Marzo, et al., Science 281, 2027-2031 (1998);C. Brenner, et al., Oncogene 19, 329-336 (2000)). Accordingly, Bcl-2abolished the formation of Vpr52-96 induced channels in ANT-containinglipid bilayers. In contrast, in the same conditions Bax exacerbates theconductance of Vpr52-96-ANT channels to a mean value of 245±2S (ascompared to 190±2 for Vpr52-96-ANT without any further addition) (FIG.8C).

EXAMPLE 5

[0154] Double Test Protocol

[0155] 1. Purification of ANT. ANT is purified from rat heart andreconstituted in liposomes according to the protocol described byBrenner et al., Oncogene, 2000.

[0156] 2. Reconstitution of ANT in liposomes. ANT (0.03 mg/ml) isincorporated in liposomes composed of phosphatidylcholine andcardiolipine (PC:CL; 45:1; W: w; and 300 ng ANT per mg of lipids) in thepresence of 0.3% n-octyl-β-D-pyranoside for 2 min. at room temperature.If need be, other proteins or compounds are added at this stage duringincorporation (e.g., Bcl-2, Bax). Then, the detergent is eliminated bydialyzing, overnight, the liposomes against the buffer 10 mM HEPES, 125mM sucrose, pH7.4 at 4° C. (e.g.: about 1 l of buffer per 1 mlliposomes).

[0157] 3. Pore function test. The liposomes are charged with 1 mM of4-UMP (4methylumbelliferylphosphate) in 10 mM KCl by sonication (50 W,22 sec). The proteoliposomes are then separated on a Sephadex G25 columnto eliminate the non-encapsulated compounds, the elution being performedusing the buffer 10 mM HEPES, 125 mM sucrose, pH7.4 at room temperature.In a microplate with 96 wells. 25 μl of liposomes are put in each well.

[0158] 25 μl of product to be tested are added and incubated for 30-60min. with different compounds (e.g.: 30 min. for Vpr 52-96; 60 min. fora non-peptide compound) at room temperature. Then, alkaline phosphatase(5 U/ml) and 150 μl of the reaction buffer 10 mM HEPES, 125 mM sucrose,0.5 mM MgCl2, pH 7.4 are added. The plate is incubated for 15 min. whilebeing shaken at 37° C. to allow the enzymatic conversion of 4-UMP to4-umbelliferone (4-UM). The reaction is stopped by adding 150 μl Stopbuffer (10 mM HEPES-NaOH, 200 mM EDTA, pH 10). The fluorometricdetermination of 4-UM is performed (excitation: 365 nm; emission 450+−5nm). In each experiment, samples of non-treatment of liposomes, ofencapsulation of 4-UMP, and of maximum salting out of 4-UMP are createdand permit the results to be expressed as a percentage of salted 4-UMP.

[0159] 4 Antiport function test. The liposomes are charged with 1 mM ofATP (4-methylumbelliferylphosphate) in some 10 mM KCl by sonication (50W, 22 sec). The proteoliposomes are then separated on a Sephadex G25column to eliminate the non-encapsulated compounds, with elution beingperformed with some 10 mM HEPES buffer solution, 125 mM sucrose, pH7.4at room temperature. In a microplate with 96 wells, 25 μl of liposomesare put in each well.

[0160] 25 μl of product to be tested are added and incubated for 30-60min. (e.g.: 30 min. for Vpr 52-96; 60 min for a non-peptide compound) atroom temperature. Then, 25 μl luciferase (HS II Boerhinger kit) areadded, and the emitted light is immediately measured. The results areexpressed as a percentage by comparison to the maximum ATP exported inresponse to an addition of 400 μl the exterior of the liposomes.

[0161] 5. Note: Proteoliposomes charged with 4-UMP and KCl with theobjective of determining the pore function will freeze at −20° C. butthose charged with ATP and KCl to determine the translocator functionwill not.

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1 7 1 14 PRT Artificial Sequence Mitochondrial membrane permeabilizingpeptide 1 Lys Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys 1 5 102 12 PRT Artificial Sequence HIV-1 Vpr peptide 2 His Phe Arg Ile Gly CysArg His Ser Arg Ile Gly 1 5 10 3 13 PRT Artificial Sequence ANT-1peptide 3 Asp Arg His Lys Gln Phe Trp Arg Tyr Phe Ala Gly Asn 1 5 10 413 PRT Artificial Sequence ANT-2-derived peptide 4 Asp Lys Arg Thr GlnPhe Trp Arg Tyr Phe Ala Gly Asn 1 5 10 5 13 PRT Artificial SequenceScrambled ANT-1 peptide 5 Phe Gln Asn Tyr Trp Gly His Lys Arg Phe ArgAsp Ala 1 5 10 6 13 PRT Artificial Sequence Mutated ANT peptide 6 AspGly His Lys Gln Phe Trp Gly Tyr Phe Ala Gly Asn 1 5 10 7 13 PRTArtificial Sequence Human phosphate carrier protein peptide 7 Ser AsnMet Leu Gly Glu Glu Asn Thr Tyr Leu Trp Arg 1 5 10

What is claimed is:
 1. A method of preventing interaction of Vpr withANT comprising: (a) providing a molecule capable of preventing thebinding of full-length Vpr to ANT; and (b) contacting said molecule withan ANT fragment; wherein said molecule prevents the interaction of saidANT fragment with Vpr.
 2. The method claim 1, wherein said methodprevents channel formation in mitochondrial membranes.
 3. The methodclaim 1, wherein said method prevents permeabilization of mitochondrialmembranes.
 4. The method claim 1, wherein said method prevents celldeath.
 5. The method of claim 4, wherein said method prevents cell deathby apoptosis.
 6. The method of claim 1, wherein said molecule is Bcl-2or a fragment thereof.
 7. A method of screening for molecules thatcompete with the binding of the C-terminal moeity of Vpr to ANTcomprising: (a) providing a Vpr fragment capable of binding to ANT; (b)contacting said Vpr fragment with an ANT fragment capable of binding toVpr in the presence and absence of a test molecule; and (c) detectingthe binding of said Vpr fragment to said ANT fragment in the presenceand absence of a test molecule.
 8. The method of claim 7, wherein saidfragment comprises full-length Vpr.
 9. The method of claim 7, whereinsaid fragment comprises amino acids 52-96 of HIV-1 Vpr.
 10. A method ofscreening for molecules that mimic Vpr or Vpr fragments in its capacityto interact physically of with ANT comprising: a) providing a Vpr or Vprfragment capable of interacting with ANT, b) contacting said Vpr or Vprfragment with an ANT fragment capable of interacting with Vpr or Vprfragment in the presence of absence of a test molecule; and c) detectingthe binding of said Vpr or Vpr fragment to said ANT fragment in thepresence of absence of a test molecule.
 11. A peptidic or non-peptidicmolecule that prevents permeabilization of mitochondrial membranes,wherein said molecule prevents the binding of Vpr to ANT.
 12. A peptidicor non-peptidic molecule that causes permeabilization of mitochondrialmembranes, wherein said molecule enhances the binding of Vpr to ANT. 13.A pharmaceutical and diagnostic composition comprising a molecule ofclaim 11 or
 12. 14. A method for causing or preventing permeabilizationof mitochondrial membranes comprising administering a composition ofclaim 13 to a patient.
 15. A method of screening for genetic orepigenetic alterations in the expression or structure of the three ANTisoforms in humans comprising: (a) providing a fragment of Vpr, whereinsaid fragment is capable of binding to ANT, with a sample comprisinghuman ANT; (b) mixing said fragment with a biological sample comprisinghuman ANT; (c) mixing said fragment with a control sample comprisinghuman ANT; (d) detecting the binding of Vpr to ANT in said biologicalsample and said control sample; (e) correlating a difference in bindingwith a genetic or epigenetic alteration of ANT; and (f) optionallydetecting a difference in the ANT capacity to form channel in liposomeor in planar lipids bilayers.
 16. A method of quantifying the level ofthe three human ANT isoforms in a cell comprising: (a) mixing Vpr with abiological sample comprising ANT in an amount effective to bind to ANT;and (b) quantitating the level of binding of Vpr to ANT.
 17. A method ofscreening active molecules of interest that induce or prevent formationof a lethal pore by ANT comprising: (a) providing purified ANT inartificial lipid bilayers or liposomes; (b) contacting molecules ofinterest to be screened with said ANT; and (c) detecting lethal poreformation by measuring the release of labeled substrate.
 18. A method ofscreening active molecules of interest that inhibit the formation of alethal pore without preventing antiport function comprising: (a)providing a composition comprising purified ANT in artificial lipidbilayers or liposomes with a molecule that induces the formation of alethal pore; (b) contacting said composition in the presence or absenceof a test molecule. (c) detecting by fluorescence the presence of theantiport function; and (d) detecting by another fluorescence the testmolecule that inhibits the formation of a lethal pore.
 19. A method ofscreening active molecules of interest according to the claim 18,wherein in step a) the active molecule that induces the formation of alethal pore is Vpr, a fragment of Vpr, or a variant of Vpr.
 20. A methodof screening active molecules of interest according to claim 18, whereinin step a) the active molecule that induces the formation of a lethalpore is selected from the group comprising: atractyloside, mastoparan,terbutyl or diamide.
 21. A method of screening active molecules ofinterest according to claim 18, wherein in step a) the active moleculethat induces the formation of a lethal pore is selected from the groupof pro-apoptotic molecules of Bcl-2 family.
 22. A method of screeningactive molecules of interest according to claim 18, wherein in step a)the active molecule that induces the formation of lethal pore is a BAXmolecule selected from the group of pro-apoptotic molecules of Bcl-2family.
 23. An isolated or purified peptide having the sequence:DRHKQFWRYFAGN.